Comparison of CTX-M encoding plasmids present during the early phase of the ESBL pandemic in western Sweden

Plasmids encoding blaCTX-M genes have greatly shaped the evolution of E. coli producing extended-spectrum beta-lactamases (ESBL-E. coli) and adds to the global threat of multiresistant bacteria by promoting horizontal gene transfer (HGT). Here we screened the similarity of 47 blaCTX-M -encoding plasmids, from 45 epidemiologically unrelated and disperse ESBL-E. coli strains, isolated during the early phase (2009–2014) of the ESBL pandemic in western Sweden. Using optical DNA mapping (ODM), both similar and rare plasmids were identified. As many as 57% of the plasmids formed five ODM-plasmid groups of at least three similar plasmids per group. The most prevalent type (28%, IncIl, pMLST37) encoded blaCTX-M-15 (n = 10), blaCTX-M-3 (n = 2) or blaCTX-M-55 (n = 1). It was found in isolates of various sequence types (STs), including ST131. This could indicate ongoing local HGT as whole-genome sequencing only revealed similarities with a rarely reported, IncIl plasmid. The second most prevalent type (IncFII/FIA/FIB, F1:A2:B20) harboring blaCTX-M-27, was detected in ST131-C1-M27 isolates, and was similar to plasmids previously reported for this subclade. The results also highlight the need for local surveillance of plasmids and the importance of temporospatial epidemiological links so that detection of a prevalent plasmid is not overestimated as a potential plasmid transmission event in outbreak investigations.


ODM-method
Plasmids were linearized using CRISPR/Cas9.For targeting blaCTX-M group 1 genes crRNA with sequence 5′-CCGTCGCGATGTATTAGCGT-3′ was used and for targeting blaCTX-M group 9 genes gRNA 5′-AGAGAGCCGCCGCGATGTGC-3′) was used 1 .gRNA was obtained by mixing equimolar amounts of crRNA and tracrRNA (0.5 nmol each, Dharmacon Inc., Lafayette, CO, USA) in the presence of 1X NEB-3 buffer (New England Biolabs, Ipswich, MA, USA) and 1× bovine serum albumin (BSA, 0.1 µg/mL), and incubating at 4°C for 30 min.To this mixture, Cas9 protein (600 ng, Sigma Aldrich, St. Louis, MI, USA) was added and the sample was incubated at 37°C for 15 min to form Cas9-gRNA complexes.Further, plasmid DNA (60 ng) together with NEB-3 buffer and BSA was added to the tube containing the Cas9-gRNA mixture to a final volume of 30 µl.The mixture was incubated at 37°C for 1 h to let Cas9 linearize the plasmids containing the gene of interest.
In the next step the barcodes were formed by letting netropsin (Sigma-Aldrich) and YOYO-1 (Invitrogen) bind to the plasmids.Plasmid DNA was mixed with λ-DNA (48502 bp, New England Biolabs), used as size reference, YOYO-1 and netropsin in a 1.8:1:70 ratio (DNA:YOYO:netropsin) in 0.5X Tris-Borate-EDTA buffer (TBE, Sigma-Aldrich).The mixture was incubated at 50°C for 30 min.Next, sample together with Milli-Q water (total volume 500 µl) was filtered using a 3 kDa Amicon Ultra-0.5 Centrifugal Filter Unit (Millipore) in a tabletop centrifuge (Eppendorf MiniSpin®) set to 13400 rpm for 17 minutes.The filtrate was removed, and the procedure repeated two more times, the last time with 0.05X TBE with an addition of 2 % (v/v) of β-mercaptoethanol (BME, Sigma-Aldrich) to protect the DNA from photodamage during imaging.
The nanofluidic devices and the fabrication procedure are discussed in detail elsewhere 2,3 .
Briefly, the devices consist of four loading reservoirs, with microchannels connecting the reservoirs two and two.The microchannels, in turn, are connected by 200 parallel nanochannels that are 150 nm wide, 100 nm deep, and 500 µm long.The nanochannels enable stretching of the DNA molecules due to the nanoconfinement.The chip was mounted to a chuck, custommade to fit on top of an epi-fluorescence microscope (Zeiss AxioObserver.Z1).Images were collected using either a 100× oil immersion objective (Zeiss, NA = 1.46), a FITC filter (488 nm excitation/530 nm emission), and an sCMOS camera (Photometrix Prime 95B) or a 63x oil immersion objective (Zeiss, NA=1.46) with an additional 1.6x magnifier, a FITC filter (488 nm excitation/530 nm emission), and an EMCCD camera (Photometrix evolve 512x512 pixels).
The chuck was designed with one pressure inlet to each of the four reservoirs, enabling pressure-driven flow of the DNA molecules.
During an experiment, the sample is loaded in one reservoir while the other reservoirs are loaded with buffer.Using pressure, DNA is moved through one of the microchannels and concentrated at the entrance of the nanochannels.With a short pulse of higher pressure, DNA is pushed into the nanochannels where it stretches due to the confinement.An image containing 20 frames (100 ms each) is collected before the DNA is flushed out and a new set of DNA molecules are pushed into the nanochannels.
The images are analyzed using custom-made MATLAB scripts 1 .Each DNA molecule is detected, its length (in µm) is determined and the intensity variation along the molecule obtained.Images for DNA molecules of similar length are grouped together, and the intensity patterns are compared.Identical cut-site among several molecules confirm Cas9 restriction and hence the presence of the resistance gene on the plasmid.If several molecules have the same pattern and Cas9 cut-site, the average pattern is calculated and hereafter called the plasmid's barcode.λ-DNA (48 502 bp) is used as an internal size reference to retrieve the length of the plasmids in kilo base pairs (kb).
The barcodes from the different plasmids are then pairwise compared to investigate if some are similar.In this comparison we allow up to 10 % stretch of the plasmid length.If the p-value is 0.01 or lower the two barcodes are considered similar 4 .When a group of barcodes (n≥3) all have p-values that are 0.01 or lower, the barcodes/plasmids are grouped together in an ODMplasmid group.
When comparing an experimental barcode to WGS data we first create a theoretical barcode from the WGS data, with the help of a custom-made MATLAB script 5 .Then the theoretical barcode and experimental barcode are compared in the same way as described above for two experimental barcodes 6 .

Whole-genome sequencing methods
Genomic DNA was extracted as previously described according to Marmur et al 7 DNA samples were quantified with the Qubit® 2.0 fluorimeter and the QubitTM dsDNA BR kit (Thermofisher Scientific, Waltham, MA, USA).Quality was determined by analysis of ratios 260/230 and 260/280 on a NanoDrop ND-1000 spectrophotometer (Thermofisher Scientific, Waltham, MA, USA).Estimation of the distribution of DNA fragment sizes was performed, using a TapeStation 2200 (Agilent Technologies, Santa Clara, CA, USA).DNA samples were sequenced as an external service, using an Illumina NovaSeq 6000 S4 (read mode 2x150 bp) (Eurofins Genomics, Germany).In-house MinION Mk101B long read sequencing (Oxford Nanopore Technologies, United Kingdom) was performed, using a rapid barcoding sequencing kit (SQK-RBK004) and a FLOW-MIN106 vR9.4,during 72 hours on MinKNOWN software v4.3.12 (Oxford Nanopore Technologies, United Kingdom).The reads were thereafter base-called and demultiplexed using Guppy v6.0.1 (Oxford Nanopore Technologies, United Kingdom) and the quality was determined using NanoPlot v1.32.1 8 .Hybrid assembly of Illumina and nanopore data was performed using Unicycler v0.4.8 9 .Basic quality parameters were determined with the Quality Assessment Tool (QUAST) 10 .The genomes were annotated with the Prokaryotic Genome Annotation Pipeline (PGAP) for submission to GenBank 11 .

Conjugation of plasmids
The strains carrying the blaCTX-M-15 and blaCTX-M-27 plasmids 15-1 and 27-1 served as donors and the tetracycline resistant E. coli strain CAG18439 served as recipient in filter mating assays.
Equal biomasses of the donor and recipient were mixed on 0.22 µm filters placed on Mueller-Hinton agar (MHA).Filters with the respective donor and the recipient alone were prepared as controls.After incubating at 37°C overnight, MHA supplemented with either tetracycline plus cefotaxime or with tetracycline alone was used to select for transconjugants and recipients, respectively.Retrieved transconjugants were checked for carriage of the expected CTX-M gene by PCR.

Figure S2 .
Figure S2.Comparison of the 15-1 plasmid with the theoretical barcode of the pEK204 plasmid described by Woodford et al 21 .